Regulation of Hepatitis C Virus translation by the viral internal ribosome entry site and the 3´-untranslated region

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Inauguraldissertation

zur Erlangung der Doktorwürde

der Naturwissenschaftlichen Fachbereiche

Biologie, Chemie und Geowissenschaften

im Fachbereich Biologie

der Justus-Liebig-Universität Giessen

Vorgelegt von

Yutong Song

M.Sc.-Virology

Biochemisches Institut

der Medizinischen Fakultät

der Justus-Liebig-Universität Giessen

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Regulation of Hepatitis C Virus translation

by the viral internal ribosome entry site

and the 3´-untranslated region

Supervisors:

Prof. Dr. Albrecht Bindereif

Institute of Biochemistry

Faculty

of

Biology

Justus-Liebig-University-Giessen

HDoz.

Dr.

Michael

Niepmann

Institute of Biochemistry

Faculty

of

Medicine

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This work was accomplished from December 2001 to November 2005 under the supervision of HDoz. Dr. Michael Niepmann in the group of Prof. Dr. Ewald Beck in the Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen.

Acknowledgements

I would like to specially thank my direct supervisor HDoz. Dr. Michael Niepmann for his constant support, excellent guidance and creative discussions which were the key to my success on the study. Moreover, it is his encouragement and valuable suggestions that gave me motives and inspiration to make progress in my scientific research.

I would like to express my sincere thanks to Prof. Dr. Ewald Beck who has also invested a great effort in my work for his support and scientific supervision. Simultaneously, I am greatly indebted to Charlotte Beck for her personal care and kind support to my family.

Further I would like to thank Prof. Dr. Albrecht Bindereif for the co-supervision of this work and for his instructive advice and his great help on my study, as well as for his cooperation in the hnRNP L project. I would like to thank my colleagues who create together such a nice atmosphere in the lab: Eleni Tzima, Christiane Jünemann, Barbara Preiss, Ralf Füllkrug, Michael Heimann, Pilar Hernández-Pastor, Dajana Henschker, Jochen Wiesner, Martin Hintz, René Röhrich, Nadine Englert, Hassan Jomaa and also other colleagues in the research groups of Prof. Dr. K. T. Preissner and Prof. Dr. R. Geyer at the Institute of Biochemistry. I express also my special thanks to my former colleagues Dr. Gergis Bassili and Amandus Zeller who were always ready to share their experiences with me, and often created an amicable occasion via their distinctive hearty spirit in our group. I have learned a lot from them at the beginning of my Ph.D. study. My sincere thanks also go to my former colleagues: Dr. Ann-Kristin Kollas, Dr. Boran Altincicek, and Frau Ursula Jost.

Also I want to thank Dr. Dieter Glebe, Dr. Michael Kann and Dr. Sandip Kanse who generously provided to me some mammalian cell lines (mentioned in section 2.1 Materials) and Silke Schreiner for the preparation of hnRNP L protein. I would like to thank my Chinese friends Jingyi Hui and Wenjun Ma for their kind help during my work.

I offer my thanks to the following academic programs that helped me to be in touch with life science, the Ph.D. Program (Graduiertenkolleg) "Biochemie von Nukleoproteinkomplexen" and the Sonderforschungs-bereich 535 (Collaborative Research Center) "Invasionsmechanismen und Replikationsstrategien von Krankheitserregern", which are founded by the Deutsche Forschungsgemeinschaft (DFG, German National Science Foundation) for the financial support.

I am forever indebted to my mother and my younger sister for providing me a sustained understanding and encouragement. Finally, I am forever grateful to my beloved wife for her love with great cordiality, her quietly unselfish support and her meticulous cares which are always with me whenever I need.

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I Zusammenfassung

In dieser Arbeit wurde die Regulation der Translation des Hepatitis C Virus (HCV) durch die Interne Ribosomen-Eintrittsstelle (IRES) und die 3´-untranslatierte Region (3´-UTR) untersucht. Die 3´-UTR stimuliert die Translation, und einige bekannte zelluläre RNA-bindende Proteine wie auch ein neu entdecktes 210 kDa-Protein binden an die 3´-UTR und sind möglicherweise an der Regulation der Translation von HCV beteiligt.

HCV, der Erreger der non-A, non-B-Hepatitis (NANBH), ist einziger Vertreter des Genus Hepacivirus in der Familie Flaviviridae. HCV hat mehr als 170 Millionen Menschen infiziert. Etwa 80 % von ihnen sind nicht in der Lage, das Virus zu eliminieren, und tragen ein hohes Risiko, chronische Leberkrankheiten wie Zirrhose und Hepatozelluläres Karzinom zu entwickeln. Ein seit kurzem verfügbares Replikon-System hat die HCV-Forschung stark beschleunigt, aber es gibt noch kein Zellkultursystem, das einen kompletten Infektionszyklus von HCV erlaubt, ein Umstand, der die Untersuchung des viralen Lebenszyklus wie auch die Entwicklung von Impfstoffen und Medikamenten noch erheblich verlangsamt.

In dieser Arbeit wurde die Interaktion einiger bekannter zellulärer RNA-bindender Proteine, des Polypyrimidine Tract-Binding Protein (PTB), des heterogeneous nuclear Ribonucleoprotein L (hnRNP L) und des Proteins, das "upstream of N-ras" codiert wird (Unr), mit der HCV IRES und der 3´-UTR untersucht. PTB bindet nicht an die IRES, aber an die 3´-UTR. Im Gegensatz dazu bindet hnRNP L nur an die IRES. Darüber hinaus fördert hnRNP L die Bindung von PTB an die 3´-UTR, was darauf hindeutet, dass beide Proteine synergistisch an die HCV-RNA binden. Allerdings konnte nur eine sehr geringe Stimulation der HCV-Translation durch hnRNP L in vitro festgestellt werden. Auch rekombinantes Unr-Protein bindet an die HCV 3´-UTR, aber in den hier durchgeführten in vitro-Experimenten konnte kein signifikanter Effekt auf die Translation festgestellt werden.

Aufgrund etlicher widersprüchlicher Berichte über eine mögliche Funktion der HCV 3´-UTR bei der Translation wurde hier festgestellt, dass mehrere Aspekte der Struktur der Reporter-Konstrukte wichtige Parameter beim Test der Funktion der 3´-UTR sind. Die 3´-UTR stimuliert die Translation nur dann, wenn monocistronische Reporter-mRNAs mit einem präzisen, authentischen 3´-Ende der 3´-UTR verwendet werden. Diese Stimulation ist stärker in Zelllinien, die von Leberzellen abgeleitet sind, als in anderen Zelllinien. In der 3´-UTR sind die Variable Region, der poly(U/C)-Trakt und der am weitesten 3´-terminal gelegene Stem-Loop 1 der hoch-konservierten 3´-X-Region für die Stimulation wichtig, weniger aber die Stem-Loops 2 und 3. Die Signale für die Stimulation der Translation überlappen also zum Teil mit denen für die Initiation der RNA-Minusstrang-Synthese, so dass diese Sequenzen möglicherweise zusammen mit viralen und/oder zellulären Proteinen an einer Interaktion der 5´- und 3´-Enden des viralen Genoms und einer Umschaltung von der Translation zur RNA-Minusstrang-Synthese beteiligt sind.

Die Suche nach Proteinen, die an der Translations-Initiation von HCV beteiligt sind, ergab zunächst kein neues Protein, das an die HCV IRES bindet. Aufgrund des Befundes dieser Arbeit, dass die 3´-UTR die Translation stimuliert, wurde dann eine Regulation der Translation auch durch Proteine, die an die 3´-UTR binden, erwogen. Deshalb wurde das Design der Reporter-Konstrukte überdacht, mit dem Resultat, dass ein bisher unbekanntes Protein entdeckt wurde, das spezifisch an die HCV-RNA bindet. Dieses 210 kDa-Protein bindet an die Variable Region der UTR nur dann, wenn eine RNA mit einem authentischen 3´-Ende der 3´-UTR verwendet wird. Dies legt die Vermutung nahe, dass das 210 kDa-Protein möglicher-weise im Zusammenhang mit der Termination der Translation an die HCV-RNA oder an das Ribosom bindet und an der Umschaltung von der Translation zur RNA-Minusstrang-Synthese beteiligt ist.

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II Summary

In this study, the regulation of translation of Hepatitis C Virus (HCV) by the internal ribosome entry site (IRES) and the 3´-untranslated region (3´-UTR) was investigated. The 3´-UTR stimulates HCV IRES-directed translation, and some known cellular RNA-binding proteins as well as a newly discovered 210 kDa protein specifically binding to the 3´-UTR may be involved in HCV translation regulation.

HCV, the main causative agent of non-A, non-B hepatitis (NANBH), belongs to the unique genus

Hepacivirus in the family Flaviviridae. HCV has infected more than 170 million people worldwide, about

80 % of whom are unable to eliminate the virus, and those are at high risk to develop chronic liver diseases including cirrhosis and hepatocellular carcinoma. The recent development of replicon systems has largely accelerated HCV research, but there is still no tissue culture system supporting a complete replication cycle of HCV, a circumstance that has slowed down studies on the basic understanding of the viral life cycle as well as drug and vaccine development.

The interactions of some known cellular RNA-binding proteins, including polypyrimidine tract-binding protein (PTB), heterogeneous nuclear ribonucleoprotein L (hnRNP L) and the protein encoded upstream of

N-ras (Unr), with the HCV IRES and the 3´-UTR were examined. There is no direct interaction of PTB

with the HCV IRES, but PTB binds specifically to the 3´-UTR. In contrast, hnRNP L binds to the IRES only. In addition, the binding of PTB to the 3´-UTR can be strengthened by hnRNP L, indicating that there is a synergistic interaction between PTB and hnRNP L. However, only a very slight positive effect of hnRNP L on HCV translation was observed in vitro. The recombinant Unr protein used in this work binds to the HCV 3´-UTR, but no significant effect of Unr on HCV translation could be observed in vitro.

Considering previous conflicting reports on a possible function of the HCV 3´-UTR in translation stimulation, it was found that reporter construct design is an important parameter in experiments testing 3´-UTR function. A translation enhancer function of the HCV 3´-3´-UTR was detected only after transfection of monocistronic reporter RNAs and depends on a precise 3´-terminus of the HCV 3´-UTR. The 3´-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in non-liver cell lines. Within the 3´-UTR the variable region, the poly(U/C)-tract and the most 3´-terminal stem-loop 1 of the highly conserved 3´-X region contribute significantly to translation enhancement, whereas the stem-loops 2 and 3 of the 3´-X region are involved only to minor extents. Thus, the signals for translation enhancement and the initiation of RNA minus-strand synthesis in the HCV 3´-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3´-5´-end interaction and in a switch between translation and RNA replication.

In an initial attempt to search for trans-acting factors possibly involved in the translation initiation of HCV, no new protein was detected to bind to the HCV IRES. From the finding that the 3´-UTR stimulates translation, it was assumed that the translation initiation could be positively regulated by proteins binding to the 3´-UTR. This gave rise to a reconsideration of the experimental design of the HCV RNA constructs used for the search for new proteins, finally resulting in the discovery of a novel, yet unknown protein that binds to the HCV RNA. This unknown 210 kDa protein binds to the variable region of the HCV 3´-UTR only when a reporter RNA with exact 3´-terminus of 3´-UTR was used. This suggests that the protein may be involved in the regulation of translation stimulation by interacting, perhaps together with other yet undiscovered proteins, with the 3´-end of the HCV 3´-UTR, and the protein(s) may be involved in a switch from translation to negative-strand RNA synthesis in the life cycle of HCV.

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III Abbreviations

A Adenine

AA amino acids

Ampr Ampicillin resistant

APS Ammonium persulfate

ATP Adenosine triphosphate

BCIP 5-Bromo-4-chloro-3-indolylphosphate

BDV Borna disease virus

bp base pairs

BSA Bovine serum albumin

oC centigrade

C Cytosine

CAT chloramphenicol acetyltransferase

CMV cytomegalovirus

CSFV Classical swine fever virus

CTP Cytosine triphosphate

d deoxy-

dd dideoxy-

ddH2O double distilled water

DMEM Dulbecco's Modified Eagle's medium

DNA deoxyribonucleic acid

DNase deoxyribonuclease

dNTP deoxynucleoside triphosphate

DTT dithiothreitol

E. coli Escherichia coli

EDTA Ethylenediamine tetraacetic acid

EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N´,N-tetraacetic acid

EMCV Encephalomyocarditis virus

Et.Br Ethidium bromide

eIF eukaryotic initiation factor(s)

et al. et alii (=and others)

FBS Fetal bovin serum

FMDV Foot-and-mouth disease virus G Guanine

GTP guanosine triphosphate

HAV Hepatitis A virus

HBV Hepatitis B virus

HCV Hepatitis C virus

HDV Hepatitis delta virus

HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

His histidine

hnRNP Heterogeneous nuclear ribonucleoprotein

IFN interferon

Ig immunoglobulin IRES internal ribosome entry site

kb kilobasepairs kDa kilodalton µg microgram µl microliter µM micromolar mg milligram min minute(s) ml milliliter

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mM mmol/l M mol/l

MOI multiplicity of infection

mRNA Messenger RNA

n nano-

NBT Nitro blue tetrazolium

NCR noncoding region(s)

ng nanogram

NMD nonsense-mediated mRNA decay

NS nonstructural protein

nt nucleotide(s)

NTP nucleoside triphosphate

NTR non-translated region

OD optical density

ORF Open reading frame

p pico-

PAA polyacrylamide

PABP poly(A)-binding protein

PAGE polyacrylamide gel eletrophoresis

PBS phosphate-buffered saline

PCR polymerase chain reaction

PKR dsRNA activated protein kinase

pmol picomolar

PMSF Phenyl-methyl-sulfonyl-fluoride

PTB Polypyrimidine tract-binding protein

rpm resolution per minute

RNA ribonucleic acid

RNase Ribonuclease

RNPs ribonucleoproteins

RPA RNase protection assay

RRM RNA recognition domain

rNTP ribonucleoside triphosphat

RT room temperature

SDS sodium dodecyl sulfate

T Thymine

TCA Trichloroacetic acid

Tris tris-hydroxymethylaminomethane

tRNA transfer ribinucleic acid

U unit (Enzyme unit)

U Uracil

Unr Protein encoded upstream of N-ras

UTP uridine triphosphate

UTR untranslated region

UV ultraviolet

vol volume

v/v volume/volume

w/v weight/volume

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Contents

Acknowledgements

I Zusammenfassung

II Summary

III Abbreviations

1 Introduction... 1

1.1 The history of non-A, non-B hepatitis ... 1

1.2 The discovery of Hepatitis C Virus... 2

1.3 The Family of Flaviviridae ... 3

1.4 The structure and genomic organization of HCV ... 4

1.5 The life cycle of Hepatitis C Virus... 8

1.6 Translation of HCV RNA is mediated by internal ribosome entry ... 11

1.6.1 Eukaryotic translation initiation... 11

1.6.2 Internal initiation of translation ... 15

1.6.2.1 General organization of viral IRES elements ... 16

1.6.2.2 The HCV internal ribosome entry site ... 17

1.6.2.2.1 The HCV 5´-untranslated region (5´-UTR) ... 17

1.6.2.2.2 Detection of an internal ribosome entry site element ... 18

1.6.2.2.3 Structural features of the HCV IRES... 20

1.6.2.3 Only eIF2 and eIF3 are required for translation initiation of HCV RNA ... 20

1.6.2.4 Cellular trans-acting factors interacting with HCV IRES ... 22

1.6.3 The HCV 3´-untranslated region (3´-UTR) ... 24

1.7 Aims of this work... 25

2 Materials and Methods... 26

2.1 Materials ... 26

2.1.1 Bacterial strains and cell lines ... 26

2.1.1.1 Bacterial strains ... 26

2.1.1.2 Mammalian cell lines ... 26

2.1.2 Materials for bacterial growth and cell culture ... 26

2.1.2.1 Materials for bacterial growth ... 26

2.1.2.2 Materials for cell culture ... 27

2.1.3 Plasmids ... 27

2.1.4 Oligonucleotides ... 27

2.1.5 Enzymes ... 29

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2.1.5.2 Modifying enzymes ... 29

2.1.6 Nucleotides... 30

2.1.6.1 Radioactive nucleotides... 30

2.1.6.2 Non-radioactive nucleotides ... 30

2.1.7 Size markers ... 30

2.1.7.1 Protein size markers... 30

2.1.7.2 DNA size markers ... 31

2.1.8 Recombinant proteins... 31

2.1.9 Chemicals and reagents... 31

2.1.10 Kits ... 32

2.1.11 Cell culture flasks and pipets ... 32

2.1.12 Photo materials and X-ray films... 32

2.1.13 Equipments... 32

2.1.14 Buffers and solutions... 33

2.1.14.1 Buffers for DNA and RNA gel electrophoresis ... 33

2.1.14.2 Buffers for protein gel electrophoresis ... 33

2.1.14.3 Buffers for molecular biological methods ... 34

2.1.14.4 Buffer for plasmid DNA preparations ... 34

2.1.14.5 Buffers and solutions for DNA purification with affinity columns ... 35

2.1.14.6 Buffers for immunological methods ... 35

2.1.14.7 Protein-RNA interaction buffers... 36

2.1.14.8 Buffers for preparation of S10 lysate of HeLa or Huh-7 cells... 36

2.1.14.9 Immunoprecipitation buffers ... 37

2.1.14.10 Buffers for in vitro translation ... 37

2.15.4.11 Buffers for purification of His6-tagged proteins under native conditions (Ni-NTA)... 38

2.1.14.12 Buffers for RNase Protection Assay (RPA)... 38

2.2 Methods... 39

2.2.1 Microbiolobical methods ... 39

2.2.1.1 Preparation of competent bacterial cells -- Classical CaCl2 method... 39

2.2.1.2 Transformation of competent cells ... 39

2.2.2 Molecular biological methods ... 39

2.2.2.1 Preparation of plasmid DNA ... 39

2.2.2.2 Enzymatic modifications of DNA ... 41

2.2.2.3 Proteinase K digestion ... 42

2.2.2.4 The polymerase chain reaction (PCR) ... 42

2.2.3 In vitro transcription and translation... 43

2.2.3.1 Preparation of DNA templates for in vitro RNA transcription ... 43

2.2.3.2 In vitro transcription with T7- or SP6-RNA polymerase ... 43

2.2.3.3 In vitro transcription of radio-labelled RNA... 44

2.2.3.4 In vitro translation... 44

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2.2.4.1 Capping of in vitro transcribed RNA... 45

2.2.4.2 Poly(A) tailing of in vitro transcribed RNA ... 45

2.2.5 Detection of reporter gene ... 46

2.2.5.1 Detection of Firefly luciferase (FLuc) reporter gene ... 46

2.2.5.2 Detection of Renilla luciferase (RLuc) reporter gene... 47

2.2.6 RNA-protein interactions... 47

2.2.6.1 UV cross-linking reaction ... 47

2.2.6.2 Electrophoretic mobility shift assay (EMSA)... 48

2.2.7 Biochemical methods... 49

2.2.7.1 Purification of recombinant proteins by Ni-NTA-His-tag-protein ... 49

2.2.7.2 Depletion of PTB from rabbit reticulocyte lysate (RRL) ... 49

2.2.7.3 Preparation of S10 cytoplasmic lysates from HeLa or Huh-7 cells ... 49

2.2.8 Immunological methods ... 50

2.2.8.1 Western blot... 50

2.2.8.2 Immunoprecipitation (IP) ... 50

2.2.9 Gel electrophoresis ... 51

2.2.9.1 Agarose gel electrophoresis and recovery of DNA fragments from agarose gels ... 51

2.2.9.2 Denaturing polyacrylamide gel electrophoresis... 51

2.2.9.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ... 52

2.2.9.4 Coomassie brilliant blue staining... 52

2.2.9.5 Autoradiography ... 52

2.2.10 Cell culture methods ... 52

2.2.10.1 Subculture protocol of adherent cell lines ... 52

2.2.10.2 Freezing protocol of mammalian cells... 53

2.2.10.3 Resuscitation of frozen cells ... 53

2.2.11 Transfection of nucleic acids into mammalian cell cultures... 53

2.2.11.1 Transfection with DNA (Lipofectamine 2000 method)... 53

2.2.11.2 Transfection with RNA... 53

2.2.12 Ribonuclease protection assay (RPA)... 54

2.2.12.1 Synthesis of [α-32 P]-labelled RNA probe and purification of the probe ... 54

2.2.12.2 Preparation of sample RNA ... 55

2.2.12.3 Hybridization and RNase digestion of probe and sample RNA ... 55

2.2.12.4 Separation and detection of protected fragments... 55

3 Results ... 56

3.1 Part I: The search for unknown cellular proteins which interact with the Hepatitis C Virus 5´- and 3´-untranslated region... 56

3.1.1 Optimization of protein binding to the HCV IRES RNA ... 57

3.1.2 Analysis of the interaction of proteins from cellular lysates with the HCV IRES and its deletion mutants... 59

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3.1.4 Choice of the optimal radioactive-labelling ribonucleotide

for the detection of proteins binding to the HCV RNA... 63

3.1.5 Optimization of protein binding specificity ... 65

3.1.6 Detection of proteins in ammonium sulfate precipitation fractions ... 68

3.1.7 Conclusions... 70

3.2 Part II: Synergetic interaction of known cellular proteins – PTB, hnRNP L and Unr – with the Hepatitis C Virus RNA ... 71

3.2.1 Interaction of polypyrimidine tract-binding protein (PTB) with HCV RNAs ... 71

3.2.1.1 Interaction of recombinant and cellular PTB with different HCV RNAs analyzed by UV cross-linking assay ... 71

3.2.1.2 Effect of PTB on HCV translation in reticulocyte lysate ... 73

3.2.2 Interaction of heterogeneous nuclear ribonucleoprotein L (hnRNP L) with HCV RNAs... 77

3.2.2.1 Detection of the interaction of hnRNP L with different HCV RNAs by shifts and UV cross-linking assay ... 78

3.2.2.2 Effect of recombinant hnRNP L on HCV translation ... 81

3.2.3 Interaction of the protein encoded upstream of N-ras (Unr) with HCV RNAs ... 82

3.2.3.1 Interaction of recombinant Unr with different HCV RNAs by gel shift assay and UV cross-linking assay ... 83

3.2.3.2 Effect of recombinant Unr on HCV translation ... 85

3.2.4 Interaction of PTB, hnRNP L and Unr with HCV RNAs ... 85

3.2.4.1 Interaction of PTB, hnRNP L and Unr in concert with the HCV RNAs... 86

3.2.4.1.1 PTB and hnRNP L... 86

3.2.4.1.2 Unr and hnRNP L... 88

3.2.4.1.3 PTB and Unr ... 89

3.2.4.1.4 PTB, hnRNP L and Unr ... 90

3.2.4.2 Effects of PTB, hnRNP L and Unr on the HCV translation... 91

3.2.5 Conclusions ... 92

3.3 Part III: The Hepatitis C Virus RNA 3´-untranslated region strongly enhances translation directed by its internal ribosomal entry site ... 93

3.3.1 The reporter construct used to analyze the possible influence of the HCV 3´-UTR on translation ... 93

3.3.2 Optimization of in vitro translation conditions ... 94

3.3.3 Effect of the HCV 3´-UTR on IRES-mediated translation in rabbit reticulocyte lysate .... 96

3.3.4 Influence of reporter construct design and transfection protocol details detection of translation enhancement by the 3´-UTR... 97

3.3.5 Expression differences are not due to differences in RNA stability or transfection efficiency ... 99

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3.3.7 The HCV 3´-UTR enhances IRES-dependent translation

preferentially in human liver-derived cell lines... 103

3.3.8 Interaction of the protein encoded upstream of N-ras (Unr) with HCV RNAs ... 104

3.3.9 Conclusions ... 106

3.4 Part IV: An unknown protein of about 210 kDa binds to the HCV RNA only in the presence of both 5´- and 3´-UTR ... 107

3.4.1 Redesigning the HCV RNA constructs used for the protein research... 107

3.4.2 Detection of an unknown protein binding to the HCV RNA with a precise end of the 3´-UTR ... 108

3.4.3 The unknown protein interacts with the variable region of the HCV 3´-UTR... 112

3.4.4 Conclusions ... 115

4 Discussion... 116

4.1 Interaction of cellular proteins with the HCV RNA untranslated regions and their effects ... 116

4.1.1 PTB ... 116

4.1.2 hnRNP L protein ... 119

4.1.3 Unr protein ... 121

4.2 The HCV 3´-UTR strongly enhances the IRES-dependent translation... 122

4.3 Regulation of HCV translation by a novel, as yet unknown 210 kDa protein? ... 128

5 References ... 131

Appendices... 147

pHCV wt clone map... 147

Interaction of FSAP with HCV RNA ... 148

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1 Introduction

Viruses (from the latin "virus", meaning "poison") are small infectious particles that consist of proteins and only one type of nucleic acids, either DNA or RNA, which is packaged in a protein capsid. In 1898, Friedrich Loeffler and Paul Frosch found that the causative agent of foot-and-mouth disease in livestock is an infectious particle smaller than any bacterium. This was the first clue to the nature of the viruses (Loeffler & Frosch, 1964). In order to replicate, viruses must infect a suitable host cell since they lack most of the internal structure and machinery which characterize "life", including the biosynthetic machinery that is necessary for reproduction. Viruses infect bacteria (then the virus is called a bacteriophage), plants, animals and humans, and they are the cause of a very wide range of human diseases like the common cold, hepatitis, AIDS, smallpox, flu, poliomyelitis, the lethal haemorrhagic disease caused by ebolaviruses, and the latest disease of severe acute respiratory syndrome (SARS) which was first recognized in March 2003 and subsequently found to be caused by a new type of coronavirus (SARS-CoV) (WHO data).

The present study focuses on the internal initiation of translation, a characteristic mechanism of translation used by Hepatitis C Virus (Family: Flaviviridae, Genus: Hepacivirus) and foot-and-mouth disease virus (Family:

Picornaviridae) to initiate their own polyprotein synthesis. Some cis- and trans-acting factors possibly involved

in the process of HCV translation were principally highlighted.

1.1 The history of non-A, non-B hepatitis

The first evidence for the existence of a non-A, non-B (NANB) hepatitis agent came from studies of multiple attacks of viral hepatitis in narcotic addicts in the 1950s (Havens, 1956). The existence of two forms of hepatitis, ‘infectious’ (type A) and ‘serum’ (type B), had been recognized in the 1940s and further reports revealed that no patient had more than one attack of either form of hepatitis. In a study of 30 episodes of acute viral hepatitis in patients, two of 30 (7 %) were found to be caused by hepatitis A, and 12 (40 %) by hepatitis B (Mosley et al., 1977). Thus there were 16 cases (53 %) not attributable to either of the two known hepatitis viruses. Exclusion of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in these patients strengthened the argument for the existence of NANB hepatitis agents.

The discovery of "Australia antigen" in 1964, by Blumberg, and its association with viral hepatitis led to detailed studies of post-transfusion hepatitis (PTH) (Blumberg, 1964). The first studies were carried out in the 1960s, prior to the screening of blood for hepatitis B surface antigen (HBsAg), when approximately 50 % of multiply transfused patients developed PTH in the USA. Most of the available blood at that time came from commercial blood donors and the equivalent rate for PTH from voluntary blood donors was much lower with one study reporting that none of 28 patients receiving volunteer blood developed hepatitis (Walsh et al., 1970). The introduction of screening and exclusion of commercial blood donors led to a dramatic fall in PTH from 30.6 cases/1000 units to 3.7 cases/1000 units of blood transfused (Alter et al., 1972). Although it was not possible to estimate the individual effects, it was generally thought that the exclusion of the commercial donor was the most significant determinant in the decrease in PTH. However, the findings that PTH had not been eradicated by these

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measures, as well as evidence that the presence of "Australia" (HBsAg) antigen could not prevent PTH (Holland et al., 1969), led to the search for alternative hepatitis agents. In one study in the early 1970s the majority of cases of PTH following cardiac surgery were not caused by HBV, and could not be explained by infection with EBV or CMV viruses (Purcell et al., 1971). Another study revealed that 12 of 108 (11 %) prospectively followed multiply transfused cardiac surgery patients developed PTH, despite receiving volunteer blood tested for HBsAg (Alter et al., 1975). Four of these 12 patients developed hepatitis B virus (HBV) infection. The other eight cases of PTH were shown not to be related to HBV, hepatitis A virus (HAV), EBV or CMV, suggesting that NANB hepatitis was the major cause of PTH in this group of patients. Studies of hepatitis in drug addicts also identified a separate type of hepatitis, which was characterized by the absence of HBsAg and of high levels of immunoglobulin M (IgM), suggestive of HAV infection (Iwarson et al., 1973).

By the late 1970s, 7-10 % of blood transfusion recipients still developed hepatitis. Serological tests failed to implicate hepatitis A or B in 90 % of these patients and the term "non-A, non-B (NANB) hepatitis" was used to describe this condition. The tendency for this condition to become chronic with progression to cirrhosis was soon recognized (Realdi et al., 1982; Dienstag, 1983). Both infectious and non-infectious causes of NANB hepatitis had to be excluded and, in the late 1970s, transmission of the agent from humans to chimpanzees was reported (Alter et al., 1978; Tabor et al., 1978). The NANB agent appeared to be present at low levels in humans and led to chronic infection in about 50 % of chimpanzees, causing distinctive histological appearances in their livers. This agent was shown to be sensitive to organic solvents, and to pass through filters of 80 nm pore-size, suggesting that it was a small enveloped virus (Bradley et al., 1983; Bradley et al., 1985).

In the 1980s the exclusion of donors with abnormal liver function tests (LFTs) or with antibody to hepatitis B core antigen (HBcAb), two surrogate markers of NANB post-transfusion hepatitis, almost halved the rate of PTH. Despite the knowledge of a distinct PTH syndrome and evidence to show successful serial transmission of these agents, by the mid 1980s the agent responsible for NANB hepatitis remained frustratingly elusive.

1.2 The discovery of Hepatitis C Virus

Most of the early studies on NANB hepatitis had focused on chimpanzee transmission investigations, showing that the putative agent was present in most patients with NANB hepatitis at a low titre of 102-103 chimpanzee

infectious doses per ml (CID·ml-1). The agent was known to cause the appearance of distinctive, membranous

tubules within the hepatocytes of experimentally infected chimpanzees. Buoyant density studies with the tubule-forming agent suggested that it is a small enveloped RNA virus because transmission to chimpanzees could be prevented by heat, β-propiolactone, and formalin or by organic solvent inactivation of the infectious fraction (Bradley et al., 1985).

Initial studies by Choo and his colleagues at the Chiron Corporation were hampered by the inability to extract sufficiently concentrated nucleic acids from infectious material. Therefore, plasma was pooled from infected animals and, by titrating infectivity in other animals, a plasma pool was obtained with a titre of approximately 106 CID·ml-1. Following ultracentrifugation, total nucleic acid was extracted, rendered single-stranded and then

both RNA and DNA were reverse transcribed using random primers. A cDNA expression library was created by cloning the random fragments into the vector λgt11, and this was then screened with serum derived from patients diagnosed with chronic NANB hepatitis. After screening more than a million clones, one (5-1-1) was found to react with serum from several NANB hepatitis patients and also with serum from experimentally infected

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chimpanzees following the onset of hepatitis (Choo et al., 1989). Three further overlapping clones were isolated by using the 5-1-1 cDNA as a hybridization probe to screen the original library and a 1089 nucleotide continuous open-reading frame (ORF) was reconstructed. The antigen (C100-3) used in the first-generation enzyme-linked immunosorbent assay (ELISA) was prepared by expressing this ORF as a fusion polypeptide with human superoxide dismutase in yeast (Kuo et al., 1989). Most cases of NANB hepatitis were found to be associated with C100-3 antibody and this response was used to define infection with a new virus, hepatitis C virus (HCV) (Alter et al., 1989; Bruix et al., 1989; Colombo et al., 1989). Although the virus was first cloned in 1989, it was not before 1994 that there have been claims of visualization of the virus by immunoelectron microscopy (Kaito et al., 1994).

Up to date, Hepatitis C virus is the first virus detected before its viral particle is found through electron microscopy. Furthermore, the discovery of HCV is the first example achieved by way of an unprecedented use of modern molecular cloning techniques, which came to the world in the late of 1980s, rather than by the conventional method of virus isolation (Choo et al., 1989).

1.3 The Family of Flaviviridae

Based on the phylogenetic sequence comparisons, HCV appears to be more closely related to pestiviruses than to the flaviviruses. For instance, both HCV and pestiviruses contain a relatively long 5´-untranslated region (5´-UTR), or also called 5´-nontranslated region (5´-NTR), with several AUG codons, whereas flaviviruses contain a short 5´-UTR of about 95-132 nucleotides in length without an AUG (except tick-borne encephalitis virus) (Rice et al., 1986; Collett et al., 1988b; Pletnev et al., 1990). Like pestiviruses, HCV does not have a cap-structure at the 5´ terminus of the genomic RNA (Collett et al., 1989; Brock et al., 1992; Tsukiyama-Kohara et al., 1992). The translation of flaviviral RNAs is mediated by a cap-dependent scanning mechanism, and usually initiated at the 5´ end proximal AUG triplet (Chambers et al., 1990). In contrast to flaviviruses, multiple AUG triplets have been found in the long 5´-UTR of HCV and pestiviruses. The methyltransferase gene found in the flavivirus NS5 region has not been found in the HCV genome. Several experimental approaches have failed to detect a cap structure at the 5´ end of pestivirus genomic RNA (Collett et al., 1988b; Brock et al., 1992). By analogy to pestiviruses, it is likely that the HCV genomic RNA is uncapped. While several AUG codons are located upstream of the translation initiation site of the HCV polyprotein, they do not appear to function as alternative start sites (Tsukiyama-Kohara et al., 1992).

Table 1: Classification of Flaviviruses. The numbers in brackets represent the tentative strains and isolates.

(Source: International Committee Taxonomy of Viruses, 2002)

Genus Number of serotypes Members

Flavivirus 10 12(79)

Pestivirus 1 3(12)

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In the end, on the basis of several criteria including genomic organization, molecular features, and biochemical properties, HCV has been classified as the sole member of a distinct genus called Hepacivirus in the family

Flaviviridae, which includes the flaviviruses and the animal pathogenic pestiviruses (Kato et al., 1990; Choo et

al., 1991; Inchauspe et al., 1991; Okamoto et al., 1991; Takamizawa et al., 1991). Table 1 shows some fundamental information on the family of Flaviviridae.

Flaviviral virions are spherical, 40-50 nm in diameter. They are with a lipid envelope and fine peplomers that do not show any structure or symmetry. The genome consists of a single molecule of linear, positive-sense, single-stranded RNA, 10.7 kb (flaviviruses), 12.5 kb (pestiviruses), or 9.6 kb (hepatitis C virus) in size. The pestiviruses and HCV do not have cap structure at 5´-end of RNA genome, whereas the 5´-end structure of the RNA of flaviviruses consists of a type 1 cap. A type 1 cap structure (m7GpppAmp) has been found at the 5´ end

of flavivirus RNA (Chambers et al., 1990) which may have resulted from the action of the proposed methyltransferase activity in the flaviviral NS5 protein (Koonin & Dolja, 1993). Except for some tick-borne flaviviruses, the RNA does not contain a 3´-terminal poly (A) tail. Virons contain two or three membrane-associated proteins, a core protein, lipids derived from host cell membranes, and carbohydrates in the form of glycolipids and glycoproteins as well.

There are about 70 recognized diseases caused by members of the Flaviviridae family. 13 cause disease in humans, such as Yellow Fever, Dengue, and Japanese Encephalitis. Common manifestations are febrile illnesses, encephalitis, hemorraging, and hepatitis. Most of theses viruses are transmitted by mosquitoes. However, Hepatitis C is transmitted by sexual contact and blood contact like other Hepatitis B virus. It has a very narrow host range, and infects only human and chimpanzee. However, it is obviously different from the diseases of hepatitis A or hepatitis B. That is the reason why it was called non-A, non-B hepatitis (NANBH) before 1989 when Hepatitis C Virus was isolated and named. So far, HCV infects more than 170 million people worldwide and causes rising rates of liver diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The only therapy currently available is combination treatment with a high dose of interferon-α (IFN-α) and the nucleoside analogue ribavirin. However, only ~ 40% of all patients benefit from this treatment and develop a sustained response. For this reason, HCV has become a focus of intensive research worldwide since the 1990s.

1.4 The structure and genomic organization of HCV

Morphologically, the HCV viral particle has a spherical shape which is about 40-80 nm in diameter, with fine spike-like surface projections and an inner core measuring 30 to 35 nm in diameter. It consists of an envelope derived from host membranes into which are inserted the virally encoded glycoproteins surrounding an icosahedral nucleocapsid and a single-stranded RNA genome (Fig. 1). The viral capsid gives the hepatitis C virus characteristic shape and size, and protects the infectious viral RNA from ribonucleases in the environment. Unlike picornaviruses carrying a small viral protein (virion protein genome-linked; VPg) which is attached to the 5´-end through a phosphodiester linkage, HCV does not contain such a special protein. The VPg appears to play an important role in initiation of picornaviral RNA synthesis, and it is believed that this protein is cleaved off by cellular proteinases soon after the entry into infected cell. The VPg is not essential for infectivity, since full-length in vitro synthesized RNA (without VPg) is still infectious. As every enveloped virus, HCV is sensitive to detergents and dehydration.

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Envelope glycoprotein 2 Envelope

glycoprotein 1

Envelope lipid RNA genome

Capsid proteins A B Envelope glycoprotein 2 Envelope glycoprotein 1

Envelope lipid RNA genome

Capsid proteins Envelope glycoprotein 2 Envelope

glycoprotein 1

Envelope lipid RNA genome

Capsid proteins

A B

Fig. 1: Electron microscope morphology of HCV associated particles and model structure of HCV. (A) EM

photos of HCV particle. Top panels: 60-70 nm virions; lower panels: 30-40 nm putative defective interfering particles. (B) Model structure of HCV. The left side of the illustration shows the viral surface of envelope lipids and glycoproteins; the right side shows the RNA genome encased by capsid proteins.

The viral genome of HCV is represented as a single-stranded RNA molecule with positive polarity, composed of about 9600 nucleotides that are flanked by non-translated regions at the 5´- and 3´-end, respectively. The viral RNA contains only one single large open reading frame (ORF) encoding a polyprotein of 3010-3033 amino acids that is processed into individual viral proteins by both viral and cellular proteases (Fig. 2) (Selby et al., 1993; Hijikata et al., 1993b; Grakoui et al., 1993c; Bartenschlager & Lohmann, 2000a). Besides the polyprotein, the expression of a novel HCV protein has been reported (Walewski et al., 2001; Xu et al., 2001; Boulant et al., 2003). Translation of this additional viral gene product also initiates at the core gene AUG start codon, but ribosomes shift into an alternative reading frame in the vicinity of the 11th codon. The resulting 17-kDa protein is therefore called the frameshift (F) or alternative reading frame (ARF) protein (Varaklioti et al., 2002). However, the exact role of the F protein remains to be defined (Bartenschlager et al., 2004).

The HCV polyprotein is cleaved co- and post-translationally by cellular and viral proteinases into eleven different products,with the structural proteins located in the amino-terminal one-thirdand the non-structural replicative proteins in the remainder(Fig. 2). The first cleavage product of the polyproteinis the highly basic core protein, forming the major constituentof the nucleocapsid (Yasui et al., 1998). In addition, a numberof other functions like modulation of several cellular processes or induction of hepatocellular carcinoma in transgenic micehave been attributed to the core protein (Chen et al., 1997; Matsumoto et al., 1997; Chang et al., 1998; Moriya et al., 1998). Envelope proteins (E1 and E2) are highly glycosylated type 1 transmembrane proteins,forming two types of stable heterodimeric complexes: a disulfide-linkedform representing misfolded aggregates and a non-covalentlylinked heterodimer corresponding most likely to the pre-buddingcomplex (Deleersnyder et al., 1997). In addition, E2 was shownto interact with the IFN-induced double-stranded RNA-activated protein kinase PKR. Upon induction by IFN-α, this enzyme reduces protein synthesis via phosphorylation of translation initiation factor eIF2-α, but in cells containing E2, PKR is inhibited,allowing continuation of translation in the presence of IFN (Taylor et al., 1999).

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C E1 E2 2 3 4B 5A 5B

5´-UTR 3´-UTR

structural non-structural proteins

4A p7

Single-strand (+) viral RNA genome

Translation Processing E1 3 4A 5B C 2 4B 5A E2 p7 5App Hyperphosphorylation

Signal peptide peptidase Signal peptidase NS2/3 protease NS3/4A protease

F

Fig. 2: Genomic organization of HCV and processing pathways of the polyprotein. A schematic

representation of the HCV genome with the 5´- and 3´-UTR is shown in the top, the translation products are given below. Proteinases involved in processing of the polyprotein are indicated by arrows that are specified in the bottom of the figure. The major cleavage pathways leading to distinct processing intermediates, most notably E2-p7-NS2 and NS4B-5A are indicated. The hyperphosphorylation of NS5A probably occurs after full proteolytic cleavage. The F protein generated by ribosomal frameshifting is depicted above the polyprotein.

Downstream of the structural region there is a small integral membrane protein, p7, a highly hydrophobic polypeptide, which seems to function as an ion channel located at the carboxy terminusof E2 (Griffin et al., 2003; Pavlovic et al., 2003). Most of the non-structural (NS) proteins, NS2, NS3, NS4A, NS4B, NS5A and NS5B, are required for replication of the viralRNA (Lohmann et al., 1999). NS2 and the amino-terminal domain of NS3 constitute the NS2–3 proteinase, catalysing cleavageat the NS2/3 site (Hirowatari et al., 1993; Grakoui et al., 1993a; Hijikata et al., 1993a). NS3 is a bifunctional moleculecarrying, in the amino-terminal ~180 residues, a serine-typeproteinase responsible for cleavage at the NS3/4A, NS4A/B, NS4B/5Aand NS5A/B sites and, in the carboxy-terminal remainder, NTPase/helicaseactivities essential for translation and replication of theHCV genome (Eckart et al., 1993; Suzich et al., 1993; Tomei et al., 1993; Bartenschlager et al., 1993b; Grakoui et al., 1993b; Kim et al., 1995; Gwack et al., 1996; Hong et al., 1996; Tai et al., 1996; Kolykhalov et al., 2000). In addition,NS3 may have other properties involved in interference withhost cell functions like inhibition of protein kinase A-mediatedsignal transduction or cell transformation (Sakamuro et al., 1995; Borowski et al.,

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1996a). NS4A is an essential cofactor of the NS3/4A proteinase and is required for efficient polyprotein processing (Bartenschlager et al., 1994; Failla et al., 1994; Lin et al., 1994b; Tanji et al., 1995a). The function of the hydrophobic NS4B is so far unknown. The transmembranous structure of NS4B and the absence of enzymatic activities suggest that this protein might play a noncatalytic role. Presumably, its main function is the induction of membranous vesicles or membrane invaginations that may serves as scaffolds for the assembly of the HCV replication complex (Egger et al., 2002; Bartenschlager et al., 2004).

HCV proteins Molecular weight (kDa) Function

F/ARF protein 17 ?

Core 23 (precursor), 21(mature) RNA binding; nucleocapsid

E1 31-35 Envelope protein; fusion domain?

E2 70 Envelope protein; receptor binding

p7 7 Viroporin (ion channel?)

NS2 21 Component of NS2-3 proteinase

NS3 69 Component of NS2-3 and NS3/4A proteinase (N-terminal

domain); NTPase/helicase (C-terminal domain); Interference with IRF-3

NS4A 6 NS3/4A proteinase cofactor

NS4B 27 Induction of membranous vesicles

NS5A 56 and 58 (basal and

hyperphosphorylated form, respectively)

IFN-α resistance? RNA replication?

NS5B 68 RNA-dependent RNA polymerase

Table 2: HCV proteins and their presumed functions in the viral life cycle (Cited from Bartenschlager et al.,

2004).

NS5A is a fascinating protein. Many cellular proteins interact with NS5A, although their functional relevance is largely unclear (He & Katze, 2002; Tellinghuisen & Rice, 2002; Macdonald & Harris, 2004). NS5A is phosphorylated on multiple serine residues by cellular kinases and can be found in hypophosphorylated (56 kDa) and hyperphosphorylated (58 kDa) forms. Major phosphorylation sites have been determined for a few HCV isolates (Reed & Rice, 1999; Katze et al., 2000), and kinases capable of phosphorylating NS5A have been identified. These include AKT, p70S6K, MEK1, MKK6, cAMP-dependent protein kinase A-α and casein kinase II (Ide et al., 1997; Reed et al., 1997; Kim et al., 1999; Coito et al., 2004). It is not yet clear which kinases are involved in generating the different phosphoforms of NS5A, or which phosphorylation sites are functionally relevant, and the role of NS5A phosphorylation remains an area of intense interest. For the HCV-H isolate the major phosphorylationsite has been mapped to serine residue 2321 of the polyproteinand the proline-rich nature of the flanking sequence suggeststhat a proline-directed kinase is responsible for NS5A phosphorylation (Reed & Rice, 1999). The role that NS5A might play in RNA replicationis so far not known, but based on analogy with other RNA viruseswhere phosphoproteins are important regulators of replication,one could assume that NS5A plays a similar role. Apart fromsuch a function, NS5A appears to be involved in resistance ofthe infected cell to

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the antiviral effect of IFN. At least forsome HCV isolates NS5A is able to bind to PKR, blocking inhibition of translation in the IFN-treated cell (Gale et al., 1997; Gale et al., 1998c).

NS5B is the workhorse of the HCV RNA replication machinery, which has been identified as the RNA-dependent RNA polymerase (RdRp) (Behrens et al., 1996; Lohmann et al., 1997; Yuan et al., 1997; Al et al., 1998; Yamashita et al., 1998). NS5B has a typical ‘right hand’ polymerase structure, with catalytic sites in the base of the palm domain, surrounded by thumb and finger domains (Penin et al., 2004). The overall structure of NS5B is remarkably similar to the RdRP of bacteriophage Φ6 (Butcher et al., 2001). NS5B also has a low-affinity GTP-binding site, distal from the active site, which is thought to be involved in allosteric regulation of the finger–thumb interaction (Bressanelli et al., 2002). A brief summary of the individual protein products and their main function(s) is given in Table 2.

1.5 The life cycle of Hepatitis C Virus

The human liver is the main site of HCV replication, but there is strong evidence that it can also replicate in peripheral blood mononuclear cells (PBMCs) or in experimentally infected B- and T-cell lines (Esteban et al., 1998), in epithelial cells in the gut (Deforges et al., 2004) and in the central nervous system (Forton et al., 2004). Studies of viral dynamics in patients treated with IFN-alpha revealed a virion half-life of 3-5 hours and a clearance and production rate of 10-12 particles per day (Neumann et al., 1998; Zeuzem et al., 1998; Ramratnam

et al., 1999). Another feature of HCV replication is the rapid generation of virus variants, primarily due to the high error rate of the viral RNA dependent RNA polymerase (RdRp) that, based on analogies with RdRps of other plus-stranded RNA viruses, is expected to be in the range of 10-4. Indeed, HCV strains isolated from the

same individual over time (as well as from different individuals) show different biological, serological and molecular characteristics, with a significant fraction of defective virus genomes, some of which may yield defective interfering particles. Within the host, viral genomes are found as heterogeneous populations termed "quasispecies". Owing to the lack of an animal model or a cell culture system able to support efficient virus replication, our understanding of the molecular mechanisms of HCV replication comes from the study of closely related flavi- and pestiviruses. These viruses enter cells by receptor-mediated endocytosis and low-pH mediated membrane fusion in lysosomes. An idealized HCV life cycle is illustrated in Fig. 3.

Attachment and entry. The first step in a virus life cycle is the attachment of theinfectious particle to the host

cell, for which a specific interactionbetween a receptor on the cell surface and a viral attachmentprotein on the surface of the particle is required. Although the mechanism of HCV entry into cells is still unknown, the E2 glycoprotein is thought to play a major role in virus attachment to the target cell. CD81, a member of the tetraspanin superfamily of cell surface molecules and expressed on virtually all cells, is the putative receptor for HCV entry into the host cell, since this molecule strongly interacts with E2 as well as virus particles in vitro (Pileri et al., 1998). Additionally, the virus may also be able to enter the cell by binding to low-density lipoprotein (LDL) receptors (Monazahian et al., 1999). But whether interaction with the LDL receptor or CD81 leads to internalization and a productive infection remains to be determined. Based on comparison with fusion peptides of paramyxoviruses, E1 may be involved in membrane fusion (Flint et al., 1999c). The low pH within the endosomal compartment may induce a major structural rearrangement of the E1, resulting in exposure of a fusion peptide which destabilizes membranes, leading to membrane fusion and particle entry into the cytoplasm.

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ER

ER

G

ER

ER

G

Fig. 3: Hypothetical model of the HCV life cycle. Upon infection of the host cell, the plus-strand RNA genome

(+) is liberated into the cytoplasm and translated. The polyprotein is processed and viral proteins remain tightly associated with membranes of the endoplasmic reticulum (ER). Minus-strand RNA (–) is synthesized by the replicase composed of NS3 - 5B and serves as template for production of excess amounts of plus-strand RNA. Via interaction with the structural proteins plus-strand RNA is encapsidated. Particles are enveloped by budding into the lumen of the ER and virus particles are exported via transit through the Golgi (G) complex.

Polyprotein translation and processing. Following decapsidation, the genomic RNA is directly translated in

the cytoplasm. Since the genome is not capped, translation is mediated by an internal ribosome entry site (IRES) element rather than by a cap-dependent mechanism employed by most eukaryotic and cellular mRNA (this issue will be described in detail in the following part). This activity does not seem to require most of the canonical eukaryotic initiation factors (eIFs) (Pestova et al., 1998b). The very 3´ end of the HCV genome as well as several cellular factors (polypyrimidine tract-binding protein, La autoantigen, heterogeneous nuclear ribonucleoprotein L and other unidentified proteins) interact with the HCV IRES and may be also involved in translation, but this

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is still enigmatic (Ali & Siddiqui, 1995; Hahm et al., 1998b; Ito & Lai, 1999; Ali et al., 2000; Lu et al., 2004). This stimulation by host factors implies that cellular proteins that vary in abundance or activity during the cell cycle might in part regulate HCV translation (Honda et al., 2000).

Directed by the IRES, the polyprotein is translated at the rough endoplasmic reticulum (rER) and cleaved co- and post-translationally by host and viral proteinases. The individual HCV proteins form a stable replicase complex associated with intracellular membranes (Tanji et al., 1995a; Wölk et al., 2000). This allows the tight coupling of different viral functions as well as the production of viral proteins and RNA in a distinct compartment. The proteinase cofactor NS4A further contributes to efficient polyprotein cleavage and replication by increasing the metabolic stability of NS3 which is rapidly degraded in its absence, and by anchoring NS3 to intracellular membranes so that the enzyme and its substrate are concentrated in very close proximity (Hijikata et al., 1993b; Lin et al., 1997; Ishido et al., 1998; Koch & Bartenschlager, 1999; Neddermann et al., 1999).

RNA replication. The positive strand RNA genome of HCV is copied into a minus strand intermediate that

serves for synthesis of new genomic plus strands. However, the individual steps underlying RNA replication are largely unknown, even though the establishment of a tissue culture system for subgenomic replicons now facilitates the related studies (Lohmann et al., 1999). In vitro, the RNA dependent RNA polymerase (RdRp) initiates replication by elongation of a primer hybridized to an RNA homopolymer or via a "copy-back" mechanism (Behrens et al., 1996; Lohmann et al., 1997; Yuan et al., 1997; Al et al., 1998; Yamashita et al., 1998; Ferrari et al., 1999). In this latter case, sequences at the 3´ end fold back intramolecularly and hybridize, generating a 3´ end that can be used for elongation, resulting in a product about twice the length of the input template. However, in the presence of high GTP or ATP concentration, RdRp can initiate RNA synthesis de

novo, and this probably occurs in vivo (Kao et al., 1999; Oh et al., 1999; Luo et al., 2000; Zhong et al., 2000a).

Concerning the template specificity, NS5B seems to bind preferentially to a sequence in its own 3´ coding region (Cheng et al., 1999). Alternatively, the template specificity may be accomplished by the high local concentration of both NS5B and the viral RNA within the replicase complex (Lohmann et al., 1997). Efficient RNA replication very likely depends on additional viral and cellular factors, like the NS3 helicase which unwinds stable RNA structures or the phosphoprotein NS5A (Lohmann et al., 1997).

In addition to viral proteins, cellular components are probablyinvolved in RNA synthesis as well. One candidate is PTB, foundto specifically interact with sequences in the 3´-UTR (Ito & Lai, 1997; Tsuchihara et al., 1997; Chung & Kaplan, 1999). Another candidate is glyceraldehyde-3-phosphate dehydrogenase (GAPDH),binding to the poly(U)-sequence in the 3´-UTR (Petrik et al., 1999). Finally, cellular proteins provisionally called p87and p130 were identified by UV-cross linking experiments withthe X-tail sequence, but the nature of these proteins remainsto be determined (Inoue et al., 1998a).

Proteins from other viruses may also affect HCV replication.For example, in most cases, the cell lines co-infected withother viruses like human T-lymphotropic virus type I (in thecase of the MT2 T-cell line), a murine retrovirus (in the caseof the MOLT4-Ma T-cell line) or Epstein–Barr virus (EBV) (inthe case of the Daudi B-cell line) support HCVreplication (Sugawara et al., 1999). Furthermore, Sugawara etal. observed that HCV-positive patients with a hepatocellularcarcinoma frequently have a high EBV load, and presented evidencethat the enhancement of HCV replication is mediated by EBV nuclearantigen 1 (Sugawara et al., 1999).

Virion assembly and release. In the absence of systems allowing the production of sufficiently high amounts of

virus particles, the assembly of HCV cannot be studied in detail. Particle formation may be initiated by the interaction of the core protein with the 5´ half of the RNA genome (Shimoike et al., 1999). Such binding of the core protein represses translation from the IRES, suggesting a potential mechanism to switch from translation

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and replication to assembly (Zhang et al., 2002). Whether the core protein forms a distinct nucleocapsid structure or a rather non-structured ribonucleoprotein complex with the HCV genome is unknown, but the core protein is able to interact with itself (Matsumoto et al., 1996). Viral capsids acquire their envelope by budding through ER membranes, where the E1 and E2 protein are inserted (Sato et al., 1993; Dubuisson et al., 1994; Duvet et al., 1998; Cocquerel et al., 1999). Then, the virions are supposed to be exported via the constitutive secretory pathway.

1.6 Translation of HCV RNA is mediated by internal ribosome entry

Most eukaryotic and cellular mRNAs are functionally monocistronic and contain a 5´-terminal m7GpppN (where

N can be any nucleotide) cap structure on which the initiation of translation strongly depends. The initiation codon used as the start site for protein synthesis is precededby a 5´-UTR in which the length, nucleotide composition, and structure can determine the efficiency and the mechanism by which a given mRNA is translated (Hershey & Merrick, 2000). For the most part, the factors that influence the bindingof 40S subunits to the mRNA provide the limiting step in translationinitiation.

In eukaryotic cells, transcription and translation are separated in space and time. Before translation occurs in the cytoplasm, the primary transcript is modified and processed immediately in the nucleus by capping, splicing and addition of about 50 to 200 adenylic residues (poly A) at the 3´end of the mRNA. The cap structure is a 7-methylguanosine residue which is attached to the first nucleotide at the 5´end of the mRNA by an unusual 5´-to-5´ triphosphate linkage. This cap structure is essential for efficient protein synthesis, and, in addition, it contributes to the stability of the mRNA by protecting the 5´ end from phosphatases and nucleases (Palmer et al., 1993; Gunnery & Mathews, 1995). Cellular mRNAs contain a relatively short 5´-UTR of about 50 to 100 nucleotides long located between the cap structure and the open reading frame (ORF) that usually contains the authentic initiation codon (AUG). The poly (A) tail at the 3´ terminus influences both mRNA stability and translation efficiency.

1.6.1 Eukaryotic translation initiation

The translation in eukaryotes is divided into three phases: initiation, elongation and termination. The reactions in each step are promoted by soluble protein factors. The rate-limiting step of translation usually occurs during the initiation phase.

The initiation phase of translation is a complex process leading to assembly of a ribosomal 80S complex at the initiation codon of an mRNA. This process is promoted by several proteins called eukaryotic initiation factors (eIFs) (see Tables 3 and 4). Prior to initiation of translation, the 80S ribosome must dissociate into its 40S and 60S subunits. This process is facilitated by eIF3 and eIF1A which help to maintain a pool of dissociated subunits probably by an allosteric effect due to a change in the structure of the 40S subunit upon eIF3 binding (Benne & Hershey, 1976; Srivastava et al., 1992).

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eukaryotic initiation factor

Molecular weight (kDa)

Function

eIF1 12.6 AUG recognition

eIF1A 16.5 Met-tRNAi binding to 40S subunit

eIF2α eIF2β eIF2γ 36.2 39.0 51.8

affects eIF2B binding by phosphorylation binds to eIF2B, eIF5

binds GTP, Met-tRNAi , GTPase

eIF2Bα eIF2Bβ eIF2Bγ eIF2Bδ eIF2ε 33.7 39.0 50.4 57.8 80.2

nonessential, helps to recognize phosphorylated eIF2 binds GTP, helps to recognize phosphorylated eIF2 Guanine nucleotide exchange activity

binds ATP, helps to recognize phosphorylated eIF2 Guanine nucleotide exchange activity

eIF3 (see table 4) ~ 700.0 ribosomal dissociation; promotes binding of mRNA and

Met-tRNAi to 40S subunit

eIF4A I / II 44.4 / 46.3 ATP dependent RNA helicase

eIF4B 69.2 binds RNA, stimulates helicase

eIF4E 25.1 binds m7G-cap of mRNA

eIF4G I / II 171.6 / 176.5 binds eIF4E, eIF4A, eIF3, PABP·RNA

eIF5 48.9 stimulates GTPase activity of eIF2

eIF5B 139 GTPase, promotes subunit joining reaction

Table 3: Overview of the mammalian translation initiation factors (Modified from Hershey & Merrick, 2000

and Hellen & Sarnow, 2001).

Mammalian eIF3 is the largest and most complex eukaryotic translation initiation factor with an apparent molecular weight of about 700 kDa. It has been first isolated and purified from rabbit reticulocyte lysate on the bases of its ability to stimulate the translation of globin mRNA (Benne & Hershey, 1976; Safer et al., 1976). Latest results show that human eIF3 consists of at least twelve non-identical subunits (Table 4). Until now, the cDNAs encoding the subunits, p170, p116, p110, p66, p48, p47, p44, p40, p36 and p35 have been cloned and sequenced (Asano et al., 1997; Chaudhuri et al., 1997; Johnson et al., 1997; Méthot et al.,o 1997 ; Block et al., 1998). p69 that was found in 2001 has been identified as an eIF3-associated protein (Morris-Desbois et al., 2001). Yeast eIF3 contains only five subunits, TiF32, NIP1, PRT1, TIF34 and TIF35(Danaie et al., 1995; Asano et al., 1998; Phan et al., 1998; Hanachi et al., 1999) that are homologous to the human eIF3 subunits p170, p116, 110, p36 and p44, respectively(Asano et al., 1997).

The first stage in translation initiation is binding of the ternary complex eIF2·GTP·Met-tRNAi to the 40S

ribosomal subunit bound with eIF3 and eIF1A to form the 43S-preinitiation complex (Benne et al., 1978). eIF2 selects the initiator tRNA from a pool of elongator tRNAs by recognizing the methionyl residue and the A-U base pair at the end of the acceptor stem (Fig. 4).

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Human Arabidopsis Yeast

Name Mass Name Mass Name Gene Mass Comments

p170 166.5 eIF3a 114.3 p110 111.1 binds RNA

p116 98.9 eIF3b 81.9 p93 PRT1 88.1 RRM, binds eIF1, eIF5

p110 105.3 eIF3c 103.0 p90 NIP1 93.4

p66 64.0 eIF3d 66.2 RRM, binds RNA

p48 52.2 eIF3e 51.8

p47 37.6 eIF3f 31.9

p44 35.4 eIF3g 32.7 p36 TIF35 30.5 RRM, binds RNA, eIF4B

p40 39.9 eIF3h 38.4

p36 36.5 eIF3i 36.4 p39 TIF34 38.8

p35 29.0 HCR1 29.6

p28 25.1 eIF3k 25.7

p69 69.0 cell growth controlling

eIF3l 56

p135 145.2 (also called CLUI)

Table 4: eIF3 subunits from mammalian, plant and yeast cells. The human subunits are named according to

their apparent masses as determined by SDS-PAGE. The human p69 was reported recently to be an eIF3-associated protein (Morris-Desbois et al., 2001). The yeast p135 is eIF3-associated with eIF3 but has not definitely been characterized as a subunit of eIF3 (Vornlocher et al., 1999) (Modified from Hershey, 2000).

The second stage is the binding of 43S-preinitiation complex to the m7G cap structure at the 5´ end of mRNA.

The cap structure is recognized by the cap binding protein (eIF4E), a subunit of eukaryotic translation initiation factor 4F (eIF4F). Mammalian eIF4F is a heterotrimeric protein complex of 250 kDa composed of eIF4E, eIF4G and eIF4A which plays the key role in recruiting the mRNA to the 43S initiation complex (Hershey & Merrick, 2000). eIF4G is an adaptor protein which binds eIF4E, eIF4A, eIF3, and the 40S ribosome (Hentze, 1997; Sachs et al., 1997). eIF4A exhibits RNA-dependent ATPase activity (Grifo et al., 1983) and a bidirectional RNA helicase activity which is stimulated by eIF4B and eIF4H (Rozen et al., 1990; Rogers et al., 1999). The binding of eIF4F to mRNA further recruits eIF4B and eIF4H. The entire complex in conjunction with the hydrolysis of ATP is thought to melt the secondary structures near the 5´ end of the mRNA that would otherwise impair ribosome movement from the 5´ proximal region to the AUG codon (Rozen et al., 1990; Rogers et al., 1999). Following recognition of the cap structure and unwinding of the secondary structure in the 5´-UTR, the 43S preinitiation complex binds to the cap proximal region of the mRNA. This ribosomal binding is thought to be mediated by interactions of eIF4G and eIF4B with eIF3-bound 40S subunit (Lamphear et al., 1995; Méthot et al., 1996). The 40S ribosomal subunit with associated initiation factors then migrates along the 5´-UTR in 5´-3´ direction probably in a linear scanning mode until it encounters the initiation codon to form the 48S preinitiation complex (Jackson, 2000). Selection of the initiation codon most probably occurs by codon-anticodon base pairing with the Met-tRNAi. However, a few nucleotides flanking the initiation codon (–3 and +4 relative to the

A of AUG) are also important for the selection process. Purines in the context A/GCCAUGA/G are optimal for initiation. This special sequence is well-known as “Kozak sequence” (Kozak, 1989a). Both eIF1 and eIF1A

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participate in the selection of the initiation codon. In their absence, the ribosome binds to the cap structure but is not able to reach the initiation codon (Pestova et al., 1998b).

3 48S Preinitiation complex 80S complex 3 3 1A AUG Cap Cap 4B 4B 4B + GTP eIF4F Pi RNA unwinding ATP ADP + Pi GDP Pi Met 2 GTP Ternary complex Met 60S 80S Ribosome 40S 40S 40S 5 5 5 5B 5B 1 1 Cap AUG Cap AUG AUG mRNA 1A Cap 4G 4A 4E 4G 4A 4E 4G 4A 4E 4G 4A 4E 4B 1A 1 AUG 1A 1 3 40S 1A 43S Preinitiation complex 3 40S 1A 1 Met Met Met Met 60S 2 GTP 2 GTP 2 GTP GDP 2 2 GTP

Fig. 4: Schematic representation of the translation initiation pathway in eukaryotic cells (Modified from

Hershey & Merrick, 2000).

The last stage of translation initiation is the displacement of initiation factors from the 48S complex and the joining of the 60S subunit in a way that the Met-tRNAi is positioned in the P site of the ribosome to form an 80S

complex at the initiation codon ready to commence the translation of the coding sequence. This step is aided by eIF5 and eIF5B (Hershey & Merrick, 2000; Pestova et al., 2000). eIF5 promotes the hydrolysis of the GTP-bound eIF2 in the presence of the 40S subunit. The binary complex of GDP-GTP-bound eIF2 is released from the ribosome together with other initiation factors. The continuity of initiation events requires the regeneration of GTP-bound eIF2, which is stimulated by the guanine-nucleotide exchange factor (GEF), eIF2B. Liberated

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